Elucidating the molecular pathways and mechanisms regulating pharyngeal arch development is fundamental to understanding the pathogenesis of the numerous congenital syndromes involving craniofacial and cardiac structures. Pharyngeal arches harbor distinct compartments of endodermal, mesodermal and neural crest-derived cells, each participating in one another's development, yet contributing to independent lineages and morphologic structures. Heterozygous microdeletions of chromosome 22q11 are the most common microdeletion in humans and are characterized by defects in derivatives of the third and fourth pharyngeal arch and pouches, including craniofacial, aortic arch and cardiac outflow tract defects. This deletion syndrome provides an entry into understanding the molecular pathways regulating pharyngeal arch and cardiac outflow tract development. The T-box transcription factor, Tbx1, appears to be a major genetic determinant in the 22q11 locus that may contribute to some features of 22q11 deletion syndrome. Tbx1 is expressed in the pharyngeal endoderm and pharyngeal mesoderm but not the pharyngeal neural crest. Mice lacking Tbx1 have aortic arch patterning defects and other pharyngeal arch-derived defects. Some aspects of the Tbx1 mutant phenotype may be from cell autonomous effects but others that affect neural crest cells are likely to be non-cell autonomous. The cell lineages that are responsible for the observed defects and the signals that affect neural crest cells remain unknown. Therefore, we are generating mice lacking Tbx1 in specific cell lineages to dissect the mechanisms through which Tbx1 controls pharyngeal and cardiac development. In addition, we have found separable cis elements that regulate pharyngeal mesoderm and endoderm expression of Tbx1 in transgenic mice and reveal Tbx1 expression in the cardiac outflow tract. Finally, we have found that Tbx1 physically interacts with two interesting transcription factors, GATA3 and Msx1. GATA3 maps to a second DiGeorge locus on chromosome 10p12 and is expressed in the pharyngeal endoderm and Msx1 mutation causes craniofacial anomalies. We hypothesize that Tbx1 is regulated by independent factors in the pharyngeal endoderm and mesoderm and that Tbx1 has distinct functions in the endoderm and mesoderm through interactions with other tissue-specific transcription factors. The specific aims of this proposal are: 1) To determine the modular mechanism of Tbx1 gene regulation in transgenic mice; 2) To test the relative contributions of Tbx1 function in the pharyngeal endoderm, mesoderm or cardiac outflow tract during embryonic development; and 3) To understand the mechanisms through which Tbx1 regulates downstream target genes by studying its interaction with the transcription factors GATA3 and Msx1. These studies will reveal the upstream and downstream mechanisms through which Tbx1 functions and are essential to understanding the pathogenesis of syndromic types of pharyngeal arch and cardiac outflow tract defects.